1. Field of the Invention
This invention relates to a method for the radioimmunoassay of thyroid-stimulating hormone.
2. Description of the Prior Art
Double antibody radioimmunoassay principles are described by Kirkham and Hunter.sup.1. In general, these assays involve the use of a second antibody (anti-first antibody) to precipitate the first antibody-antigen complex. This method relies on the fact that the antigenic sites of the first antibody are distinct from the sites involved in the antibody activity of the molecule. In the specific case of a thyroid-stimulating hormone (TSH) assay, antibodies against TSH produced in rabbits (rabbit anti-human TSH) are first incubated with the TSH present in the aqueous sample to be determined and then with radioactively labeled TSH, so that some labeled and unlabeled TSH becomes bound, on a competitive basis, to the antibody molecules, while the remainder of unlabeled and labeled TSH remains free in solution. Second antibody (antibody against rabbit antibody molecules produced in goats) is then added, and it becomes bound to the first antibody-antigen complex. When the proportions of second antibody and first antibody are correct, a lattice of first antibody and second antibody molecules will form, resulting in a precipitate. The sample can be treated so as to effect a separation of bound labeled and unlabeled TSH from free labeled and unlabeled TSH, and the radioactivity of the bound TSH is measured. The amount of radioactivity bound is a function of the amount of unlabeled TSH present in the sample. Double antibody procedures to separate bound TSH from free hormone are available commercially. Beckman, in its radioimmunoassay for human thyroid-stimulating hormone makes use of the routine double antibody procedure to determine TSH. The procedure involves the addition of a carefully calibrated amount of a second antibody solution to form a precipitate comprising first antibody-antigen complex bound to the second antibody. FNT .sup.1 Kirkham, E. E. and Hunter, W. M., Editors, Radioimmunoassay Methods (Edinburgh, Scotland: Churchill Livingstone, 1973).
Sorin has made commercially available a solid-phase second antibody radioimmunoassay for TSH in which the second antibody is coupled to cellulose. In the Sorin assay the sample containing the TSH to be determined is mixed with radioactively labeled TSH and the first antibody at room temperature for 18-24 hours. The solid phase second antibody is then added and incubated for 3 hours with agitation. The solid phase is separated, washed two times and the radioactivity in the solid phase is measured. A radioimmunoassay for TSH which utilizes the double antibody procedure wherein the second antibody is coupled to cellulose, is also described by W. J. Sluiter et al. in Clin. Clim. Acta., 42 (1972) 255-262.
U.S. Pat. No. 3,555,143 issued Jan. 12, 1971 to R.E.A.C. Axen, et al. teaches the use of a solid phase first antibody in radioimmunoassays for proteins and polypeptides. Pharmacia has applied this single antibody technique in its commercially offered radioimmunoassay for TSH. In this procedure, the first antibody is covalently bound to a polydextran. The sample to be determined and the solid-phase first antibody are incubated at room temperature for 18-24 hours followed by addition of radioactively labeled TSH and further incubation with agitation for 18-24 hours. The solid phase is separated, washed three times and its radioactivity measured.
The patent application by Monthony et al. Ser. No. 621,197 filed Oct. 9, 1975 describes an immunofluorescent assay method in which the immune reactants are covalently bound to water insoluble hydrophilic polymeric particles of about 0.1-10 microns in size. The solid phase antibody is mixed with the antigen (or hapten) to be determined and corresponding antigen (or hapten) which has been fluorescently labeled, so as to bind a quantity of labeled and unlabeled antigen. The solid phase is separated and the fluorescence measured by optical spectroscopy, the concentration of the unknown immune reactant being a function of the value of the fluorescence.
The patent application by Wegfahrt et al., Ser. No. 718,308, filed Aug. 27, 1976 describes an improved radioimmunoassay for thyroid hormone utilizing a single antibody technique in which the antibody is covalently bound to hydrolyzed polyacrylamide particles as in Monthony et al. so as to separate bound thyroid hormone to be determined.